Values for serotonin as an indicator for the dogs emotional state Case study on eight dogs Valorile serotoninei ca indicator de apreciere a stării emoționale a câinilor Studiu pe opt cazuri Timea Andrea Kocis (Bochiș)*, Astrid-Simone Groszler, I. Țibru Universitatea de Științe Agricole și Medicină Veterinară a Banatului "Regele Mihai I al României" din Timișoara Facultatea de Medicină Veterinară dea16_tim@yahoo.com Abstract Key words: dog, serotonin, behaviour. Cuvinte cheie: câine, serotonină, comportament. The study was carried out on 8 mixt-breed dogs, ages 1-8 years old, which we took blood samples from in order to determine the serotonin content at four differnet moments. The first moment of blood sampling was registered as moment zero (M0), five minutes after fitting the cannula. At moment 1 (M1), samples were collected, then the dog experienced a state of well-being, either because the owner played with it, or because it was offered its favorite treat. Moment 2 (M2) was the moment when the dog experienced a state of anxiety becoming irritable and even aggressive, depending on its temper. At the last moment (M3), the dog was left to calm down and then a blood sample was collected. After analyzing the results obtained, it can be said that there was a positive correlation between the dog's posture and changes in serotonin values, depending on the individual's involvement. Rezumat S-au luat în studiu 8 câini, metiși, cu vârste cuprinse între 1 și 8 ani, de la care s-au recoltat probe de sânge pentru determinarea serotoninei în patru moment diferite. Primul moment în care s-a recoltat sângele a fost considerat momentul zero (M0), la interval de cinci minute după montarea branulei. În momentul 1 (M1), s-au recoltat probe, după ce câinelui i s-a indus o stare de bine, de plăcere, moment realizat fie prin faptul că proprietarul s-a jucat cu câinele sau că i s-a oferit recompensa preferată. Momentul 2 (M2) a fost momentul în care câinele a fost pus într-o situație de agitare devenind irascibil până la agresiv, în funcție de temperamentul acestuia. În ultimul moment (M3), câinele a fost lăsat să se liniştească după care s-a recoltat o nouă probă de sânge. În urma analizei rezultatelor obținute, se poate spune faptul că a existat o corelație pozitivă între postura câinelui și modificările valorilor serotoninei, în funcție de gradul de implicare al fiecărui individ în parte. Introduction Statistics show that more than one million people aer annually bitten by dogs worldwide, representing about 1% of medical emergency cases, but it is estimated that this number only represents half such events, the rest not being reported. The same statistic shows that half of the victims are children under 10 (Leon et al., 2008). Even if most wounds resulting from dog bites are minor, this does not mean that the phenomenon should be ignored or minimalized. Aggressive behaviour in dogs has a negative impact on public health and safety, the number of bitten persons increasing yearly, as well as on the animals wellbeing, aggressiveness representing the most frequent cause of dog abandonment and euthanasia. Related to aggressiveness, one may claim that there are some breeds prone to displaying such behaviour, while other breeds are considered non-aggressive (Overall, 1997). Epigenetic factors may also be involved in neurophysiological differences at the serotonin level within the same species. 60
Environmental factors, the owner s personality, the education the dog has received, are also factors directly influencing the different shaping of every individual s personality. Some authors of specialty literature have recounted behavioural similarities between the owner and the dog s aggressive behaviour (Peachey, 1993). Aggressiveness may be based on a physio-pathologic cause that is why it is necessary to exclude these, before a behavioural diagnosis is formed (Beaver, 1999). The most common aggressiveness forms include dominance, fear and intraspecific aggressiveness. A protocol, for a correct diagnosis of canine aggressiveness, must include: the determination of the aggressiveness purpose or target, the context in which it occurred, as well as the body posture of the dog before, during and after the aggressive behaviour manifested (Houpt, 1998). At the canine species, correlations have been observed between serotonin and aggressiveness, Reisner et al. (1996) noticing the fact that individuals which manifested dominant aggressiveness, showed high serotonin values. During another study, a lot of dogs that manifested aggressiveness towards their owner, displayed high serotonin values, as compared to dogs that did not display such behaviour (Cakiroglu et al. 2007, Rosado et al, 2009). Administration of serotonin value increasing medication reduce aggressiveness (Leon et al. 2008), and diets with a reduced content of tryptophan, an amino acid which precedes serotonin, leads to aggressiveness intensification (Young et al. 1991). Other studies highlighted the role of the serotonergic system in triggering the aggressive behaviour through changes occurring at the level of serotonin receptors from various cerebral areas (Peremans et al., 2003), as well as in serotonin adherence to thrombocytes (Rosado et al., 2009), in aggressive dogs as compared to the control lot. Amat et al., (2013), studied the English cocker spaniel dogs aiming to establish whether or not the breed is more aggressive than other breeds, based on the fact that lower serotonin values have been registered with this bred. All dogs included in the study were diagnosed with emotional aggressiveness, the average serotonin values at the English cocker spaniel breed reaching 318.6 ng / ml as compared to the 852.77 ng / ml average, registered with other dog breeds, the difference being significant (P< 0,05). In another study, Leon and Rosado, compared various methods of determining peripheral serotonin, and established correlations between the serotonergic system and canine aggressiveness. Serotonin values, in aggressive dogs, were lower than those obtained in nonaggressive dogs, which suggests an inverse correlation between the serotonergic system and aggressiveness. Aggressive dogs presented lower serotonin values than non-aggressive dogs (278.5 ng/ml vs 387.4 ng / ml, P <0.01). The lowest serotonin level was determined in dogs which displayed defensive aggression (Leon and Rosado, 2012). For an innovative diagnosis and treatment approach, it is necessary to understand the biologic mechanisms of dog aggressiveness. The serotonergic system and the hypothalamus-hypophysis-suprarenal (HPA) axis are considered to play an important role in aggressiveness control. In the mammal brain, the tryptophan precedes serotonin (5-hydroxytryptamin) and modulates the neuronal information processing. Tryptophan is considered as the substance controlling several behavioural types: aggressiveness, spontaneity, food selection, stimulation, sexual behaviour and reaction to pain, more than that, serotonin is also involved in emotion control (Sainio et al., 1996). Serotonin is obtained in the organism through hydroxylation and decarboxylation of tryptophan, and direct tryptophan decarboxylation leads to tryptamin. 61
Tryptamin is present in the brain as well, but in much smaller quantity, functioning as a modulator for the serotonin action on neurons (Sainio et al., 1996). 1. Material and Methods 1.1. Animals The current experiment included eight dogs, namely: two German shepherds, a German shepherd mixed-breed, three Mioritic shepherds mix-breeds, and to medium to large mixed-breeds. Each dog was set up with a cannula, in the cephalic vein and blood samples were collected at four different moments, in order to monitor the serotonin value fluctuations. The blood samples were collected in vacuum containers without anticoagulant. The first blood sampling moment was considered moment zero (M0), at a five minute interval from attaching the cannula. At moment 1 (M1), the samples were collected after the dog was induced a pleasant state of mind, either because the owner played with it or because it received its favourite treat. Moment 2 (M2) was the moment the dog was placed in an anxiety situation becoming irritable to aggressive, depending on the individual s temperament. At the last moment (M3), the dog was left to calm down, after which a new blood sample was collected. At the moment of sample collecting, each dog was filmed and photographed, in order to corroborate the momentary behaviour with the data supplied by the owner, thus we were able to describe the complete situation of the studied individuals. 1.2. The samples preparing The blood samples were stored at room temperature for two hours, then centrifuged at 3500 rotations/minute, for five minutes. The serum thus separated was collected with Pasteur pipettes and was stored in Eppendorf pipettes in the freezer, at -40 C, until the serotonin determination day. The determination of the serric serotonin was carried out with the Micron 2010 device, Version 5, the Serotonin ELISA Enzyme Immunoassay for the Quantitative Determination of Serotonin in Serum, Plasma and Urine kit, from the company DLD Diagnostika Hamburg, Germany. The determinations were carried out in the Horea Cernescu Laboratory Complex at BUASVM Timişoara, Faculty of Veterinary Medicine. 2. Results and Discussions As a result of the experiment, for each individual we determined the serotonin variations during the various moments and corroborated them with the dogs behaviour. Subject 1. Mioritic shepherd mixed-breed, male, 4 years old, used as watchdog at a cow farm. The dog was calm during the cannula set up, did not show any signs of discomfort, the first blood sample was collected at what we called M0, or reference moment. At M0, the serotonin value was of 113.55 ng / ml. M 1 represented the stage when the owner played with the dog, the dog s posture was relaxed, and the serotonin value was of 162.71 ng/ml. 62
At moment two M2, which represented the stage when the animal became aggressive, the serotonin value was of 149.19 ng / ml, While at M 3, after the animal calmed down, the serotonin value decreased to 95.10 ng / ml (fig.1). In order to register the level of individual involvement, we expressed the value dynamics in percentages, so that for dog 1 at M0, when the serotonin value was of 113.55 ng / ml, which we considered 100 %, at M1 it reached 143.29 %, at M2, la 131.38 %, and at M3, la 83.73 %. Analysing these values, we observe that at the moment the owner managed to please the dog, its involvement degree was highest, reaching 143.29 %, and during the period of discomfort and the beginning of aggressiveness manifestation, a decrease in involvement was observed, which at the end of the experiment was lower than the initial values (table 1). At the next moment, the owner interacted with the dog in an energic way, still the dog did not react, he was obedient, but the serotonin value reached 255.86 ng / ml. When continuing the activity, even brutally, the animal did not show signs of aggressiveness, but its discomfort grew visibly, reflected by a 404.30 ng / ml serotonin value (fig.1). Analysing this individual s situation, we can say that with the increase of the discomfort triggered by the owner s attitude, there was also an involvement increase, from 108.33 % to 171.18 %. We can associate these values with the dog s position at the farm (an isolated place), and the rare interaction with the owner (table 1). Subject 3 Carpathian shepherd mixed-bred, male, aged 3, aggressive, protecting its territory. Subject 2. German shepherd mixed-breed, male, under 1 year old, watchdog. At the moment the cannula was set up, it was calm, did not display any signs of discomfort, the serotonin value at M0 being of 236.18 ng / ml. It opposed the cannula setup, a firm restraining by the owner was necessary. At M0 the serotonin value was of 281.93 ng/ml. At M1, when the owner interacted with the dog, a serotonin value of 257.57 ng/ml was registered. The aggressiveness moment was triggered by a person the dog does not like, the serotonin value reaching 115.52 ng/ml. 63
At M3 the serotonin value increased even more, reaching 336.52 ng/ml (fig.1). Analysing the values registered with dog three, we observed that active presence of the owner led to a serotonin decrease to 91.35%, and the moment the dog interacted with the disagreeable person, it dropped to 40.97, ulterior reaching 119.36% (table 1). Subject 4. Carpathian shepherd mixed-breed, aged 4, female, watchdog. At M0 the serotonin value was of 583.02 ng/ml, while at the aggressiveness moment it reached 298.17 ng / ml (fig.1). Dog 5 is an individual used for watch, at the reference moment, when the first blood sample was collected, it was aggressive, and the serotonin value reaching 583.02 ng / ml, at M2 the involvement percentage was of 51.14 % (table 1). Subject 6. German shepherd, (Tesa) female, age 3. The moment of cannula setting was difficult, the dog displaying signs of anxiety and discomfort, the serotonin value at moment zero was of 128.60 ng / ml, ulterior decreasing to 183.32 ng / ml at M1. At the aggressiveness moment, manifested through barking and walking along the fence when a person was walking by in the street, the serotonin value increased to 253.76 ng / ml (fig.1). With dog 4, the serotonin value at the first moment was of 128.60 ng / ml, at M1 142.55 %, and during the aggressiveness moment 197.2 % (table 1). Subject 5. Mioritic shepherd dog mixed-breed, 5 years old, female, displays territorial aggressiveness. The serotonin value at M0 was of 188.74 ng / ml. At the next moment, when the owner played with her, the serotonin decreased to 149.74 ng / ml, and then, at the aggressiveness moment, the value dropped down to 111.73 ng / ml, at M3, it went up to 257.14 ng / ml (fig.1). Upon data analysis, we observed that at M0, the serotonin had a 188.74 ng / ml value, at M1 79.33 %, at M2, it dropped down to 59.19%, while increasing to 136.24 % during the last stage (table 1). 64
Subject 7. Subject 8. A German shepherd female (Foxy) 6 years old. The following serotonin values were registered: at moment 0, 261.3 ng / ml, at M1, the value was of 145.05 ng / ml, and at M2 181.64 ng/ml (fig.1). With this individual the percentage statistic shows that at M1 the involvement was of 55.37 %, increasing to 69.34 % (at the aggressiveness stage) (table 1). Mixed breed, (Dixie), male, 4 years old. After sampling and analyzing its blood, the following serotonin values were obtained: M0 429.30 ng/ml, M1 107.91, at the aggressiveness moment (M3) the serotonin level increased to 167.50 ng / ml (fig.1). Expressed in percentages, the situation presents itself as follows: at M1 the implication was of 25.13%, while at the moment of aggressiveness (M3) it reached 39.01% (table 1). Figure 1 Serotonin values of the owner dog group All in all, the statistical analysis (average, standard deviation and the variability coefficient) determined the following results: At M0, which represented the reference moment for each individual, the average was of 277.91, the standard deviation of 158.24 and the variability coefficient 56.94. This coefficient amounting to more than 30%, we may conclude the fact that we encounter a high variability, which can be explained by the fact that every individual showed a different reaction to the process of holding down for blood sampling. at M 1, the average was of 180.31, the standard deviaton of 5.88 and the variability coefficient 31.55%, at M 2, the average was of 182.50, the standard deviation of 69.87 and the variability coefficient 38,28, at M 3, the average was of 273.27, the standard deviation of 133.13 and the variability coefficient 48.72 %. Comparing the averages of the four work stages we can observe that in relation with M0, the average was of 277.91 ng / ml, at M1 the average decreased to 180.31ng / ml, at M2 65
when the dogs showed aggressiveness, the average was of 182.50 ng / ml, and at M3, when the owner calmed them down, the average rose to 273.27 ng / ml (table 1, fig 1). Table 1. Results of serotonin values and the involvement of the individual The analyzed moment Subject M0 M1 M2 M3 (100%) (happy) (aggressive) (calmed by owner) 1 113.55 162,71 (143.29%) 149.19 (131.38%) 95.10 (83.75%) 2 236.18 255.86 (108.33%) -- 404.30 (171.18%) 3 281.93 257.57 (91.35%) 115.52 (40.97%) 336.52 (119.36%) 4 128.60 183.32 (142.55%) 253.76 (197.32%) -- 5 583.02 -- 298.17 (51.14%) -- 6 188.74 149.74 (79.33%) 111.73 (59.19%) 257.14 (136.24%) 7 261.93 145.05 (55.37%) 181.64 (69.34%) -- 8 429.30 107.91 (25.13%) 167.50 (39.01%) -- 4. Conclusions The averages for the stages under study were M0 277.91 ng/ml; M1 180.31ng/ml; M2 when the dogs manifested aggressiveness, 182.50 ng/ml, and at M3, when they were calmed by the owner, it grew to 273.27 ng/ml. The dogs whose serotonin values varied significantle during the four moments are nervous individuals, which, following a temporary discomfort, changed their behaviour in relation with the serotonin value. The highest serotonin values occurred with three dogs: C2 (at the aggressiveness moment, 404.3 ng/ml), C5 (aggressive dog, 583.02 ng/ml), C8 (nervous dog, 429.30 ng/ml). In the case of dog 2, a young animal, 1 year old, we can conclude that, due to the lack of experience, it does not know how to manage its emotions, at the moment the owner tried to annoy it, it did not show an outward sign, but the serotonin level increased significantly. The highest serotonin value was registered in C5, an adult dog, 5 years of age, that manifested aggressiveness towards foreigners, making blood sampling difficult. Dog 8, a nervous individual, 4 years old, was shy and held back during the entire experiment, shows territorial aggressiveness. References 1. Amat Mar, Le Brech S, Camps T., Torrente C. (2013). Differences in serotonin serum concentration between aggressive English cocker spaniels and aggressive dogs of other breeds, Journal of Veterinary Behavior, 8, 19-25. 2. Beaver B.V. (1999). Canine behaviour: a guide for veterinarians, WB Saunders Company, pp. 152. 3. Cakiroglu D., Meral Y., Sancak A.A., Cifti G. (2007). Relationship between the serum concentrations of serotonin and lipids and aggression in dogs. Vet. Rec. 161, 59 61. 4. De Napoli J.S., Dodman N.H., Shuster L., Rand W.M., Gross K.L. (2000). Effect of dietary protein content and tryptophan supplementation on dominance aggression, territorial aggression, and hyperactivity in dogs. J. Am. Vet. Med. Assoc. 217, 504 508. 5. Houpt, K.A. (1998). Domestic animal behaviour for veterinarians and animal scientists, 3 rd, Ed Iowa University Press, 14-16. 6. Leon M., Rosado B., Garcia Sylvia, (2012). Assessment of serotonin in serum, plasma and platelets of aggressive dogs. Journal of Veterinary Behavior, 7, 348-352. 66
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